A dual-aptazyme system for genetic complementation reveals stage-specific roles for the <i>Trypanosoma cruzi</i> cytoskeleton-associated protein 5.5

ABSTRACT Parasitic trypanosomatids, such as Trypanosoma cruzi , Trypanosoma brucei , and Leishmania spp ., cause devastating tropical diseases, with T. cruzi being increasingly recognized as a public health concern in the United States due to established sylvatic cycles and rising autochthonous transmission. Although T. brucei has long served as the model trypanosomatid due to the array of molecular tools available for its study, T. cruzi shares greater biological similarity with the majority of trypanosomatids regarding nutrient uptake, transmission strategy, and metabolism. Recent advancements in genetic tools have now enabled in-depth studies in T. cruzi previously restricted to T. brucei . Here, using the Small Hammerhead Aptazyme-Regulated Knockdown (SHARK) system, we report the characterization of cytoskeleton-associated protein 5.5 (CAP5.5) in T. cruzi . To validate this phenotype, we introduce a novel method for simultaneous knockdown and rescue of a target protein using two tetracycline-responsive aptazymes. Knockdown of CAP5.5 revealed stage-specific roles in the maintenance of cell shape, organelle segregation, and proliferation. To quantify these phenotypes, we leveraged open-source, state-of-the-art segmentation software to develop a semi-automated, high-throughput image analysis pipeline. Our data suggest a potential role for CAP5.5 in repairing motility-related damage to the microtubule cytoskeletal array. These findings demonstrate the emerging utility of T. cruzi as a robust model trypanosomatid. IMPORTANCE Trypanosoma cruzi is the causative agent of Chagas disease, a neglected tropical disease that presents a growing public health concern. Historically, research into T. cruzi biology has been hindered by a scarcity of genetic tools, forcing scientists to rely on models from related but biologically distinct parasites. In this study, we utilize genetic tools (the SHARK conditional knockdown system) to characterize a cytoskeletal protein, CAP5.5, revealing that it is required for the parasite to maintain its shape and divide during its motile stage. We further demonstrate the use of a dual-control system that allows simultaneous gene knockdown and rescue, alongside a new high-throughput 3D imaging pipeline. This work establishes a rigorous technical framework that empowers the community to conduct in-depth investigations of essential T. cruzi genes directly, thus accelerating the discovery of potential therapeutic targets.